市售靈芝產品品管指標之研究

Study on Quality Control Markers in Commercial Ganoderma lucidum Products

高雅玲(YA-LING KAO)

指導教授 ： 許輔

國立臺灣大學/生物資源暨農學院/園藝暨景觀學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02058

摘要

本論文目的為釐清總糖、(1,3)-β-D-葡聚醣或 LZ-8 含量是否可做為代表產品活性之指標，並找到判別產品適當的活性檢測濃度範圍。第一部分測量各產品總糖含量，並對活性多醣 (1,3)-β-D-葡聚醣，以專一性之 aniline blue 螢光呈色法分析其含量。結果發現樣品間此兩種成分含量差異很大。第二部分進行活性試驗，調整樣品總糖濃度或 (1,3)-β-D-葡聚醣濃度一致時，各樣品活化小鼠腹腔巨噬細胞和巨噬細胞株 RAW 264.7 分泌 TNF-α 和 IL-6 的效果卻有顯著差異。因此，總糖與 (1,3)-β-D-葡聚醣含量不宜做為評估市售靈芝產品的活性指標。接著，將各市售靈芝產品以相同稀釋倍率與細胞共培養，結果顯示樣品能顯著活化小鼠腹腔巨噬細胞和 RAW 264.7 分泌 TNF-α 及 IL-6。進一步測試樣品稀釋倍率與活性的相關性，發現稀釋範圍介於 300-2500 μg/mL (30-250 mg 市售靈芝產品溶於 100 mL 磷酸鹽緩衝生理鹽水)，可以有效辨別各樣品是否具有活性。
　　在活化小鼠脾臟細胞的部分，市售靈芝產品皆無法顯著提升 TH1 和 TH2相關細胞激素 IFN-γ、IL-2 和 IL-4 的分泌量。此外，以膠體電泳及西方轉漬法分析，皆證實市售靈芝產品中靈芝蛋白 LZ-8 含量極低。第三部分欲以高效液相層析儀 (HPLC) 分析菌絲體中 LZ-8 含量。結果顯示，無論是否添加標準品 LZ-8 於菌絲體溶液中，皆無法偵測到 LZ-8 的存在。將菌絲體溶液通過玻璃纖維去除多醣，再添加標準品 LZ-8 後，以 HPLC 分析則可測到添加的標準品 LZ-8。因此，推測菌絲體中影響 HPLC 分析 LZ-8 含量的物質可能為多醣類。進一步以 exo-(1,3)-β-D-glucanase 和 endo-(1,3)-β-D-glucanase 切除 (1,3)-β-D-葡聚醣後，仍無法分析到菌絲體中的 LZ-8，所以僅能確定干擾分析的物質並非 (1,3)-β-D-葡聚醣，但該物質實際結構仍有待釐清。
　　綜合上述實驗結果，因總糖、(1,3)-β-D-葡聚醣和 LZ-8 皆不適合做為靈芝產品品管指標，故建議應直接分析樣品活性，可選擇巨噬細胞株 RAW 264.7 與樣品共培養後，測量 TNF-α 和 IL-6 分泌量，評估產品之活性優劣，適當產品範圍為 30-250 mg 市售靈芝產品溶於 100 mL 磷酸鹽緩衝生理鹽水，此方法可用於靈芝產品製程品管，以確保產品品質一致性。

關鍵字

(1,3)-β-D-葡聚醣 ； LZ-8 ； 靈芝多醣活性 ； 靈芝菌絲體

並列摘要

Ganoderma lucidum, a medicinal fungi, has been used in Traditional Chinese Medicine (TCM) for its health promoting constituents, including polysaccharides, proteins, triterpenes, sterols, and fatty acids. However, G. lucidum contains various and complex bioactive compounds. It was difficult to distinguish the bioactivity of commercial G. lucidum products from specific component. Most products were claimed on the basis of their total sugar or crude polysaccharides contents, but immunomodulatory protein (LZ-8) and (1,3)-β-D-glucan, were not shown in the nutrition facts. In this study, we decided to examine whether the contents of total sugar or (1,3)-β-D-glucan could be used as the quality and functional indicators, and what would be the appropriate concentrates for the evaluation in product activities. First, we determined content of total sugar and content of (1,3)-β-D-glucan in commercial products with phenol-sulfuric acid method and fluorometric method. Our results showed that there were significant differences on the contents of total sugar and (1,3)-β-D-glucan in the eight commercial products. Samples with the same contends of total sugar or (1,3)-β-D-glucan had different capability to induce secreation of TNF-α and IL-6 in murine macrophages and RAW 264.7. Therefore, the quantity of total sugar and (1,3)-β-D-glucan might not be an appropriate functional indicators. Our results also revealed that the approach of diluting the commercial product solutions which were cultured with murine macrophages to 30 to 250 mg commercial product in 100 mL PBS could be used to distinguish bioactivities of commercial products. Further, the eight commercial products could not increase production of cytokine (IFN-γ, IL-2, and IL-4) by murine splenocytes. In addition, SDS-PAGE and Western blotting analysis of these samples showed that commercial G. lucidum products could rarely include immunomodulatory protein LZ-8. In the third part, mycelia from G. lucidum were lysed by ultrasonic reactor and analyzed for the content of LZ-8 by HPLC. LZ-8 peak have been observed as lysis solution filtered through glass fiber membranes and added 100 μg/mL standard LZ-8 to the solution. As shown above, we assumed that the component which interfered the HPLC analysis of LZ-8 should be polysaccharide. Sample solution was further treated with exo-(1,3)-β-D-glucanase and endo-(1,3)-β-D-glucanase in order to remove the potented interfering substance, but no LZ-8 peak have been observed. As a result, it was inferred that the interfering substance might be polysaccharides but not (1,3)-β-D-glucans. In conclusion, the content of total sugar, (1,3)-β-D-glucan, and LZ-8 could not be the functional indicators. The appropriate dosages of sample cultured with murine macrophages were 30 to 250 mg of commercial product in 100 mL PBS and could be used to assess product activities. In the third part, we inferred that polysaccharids might be the main factor to interfer the HPLC analysis of the free form of LZ-8.

並列關鍵字

(1,3)-β-D-glucan ； LZ-8 ； Immunomodulation ； G. lucidum mycelia

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